Healthy seeds are an important component of global food security, and their microbiome was recently identified as crucial for plant growth, resilience and health. Seed vigour is highly affected by storage conditions and aging. To study the impact of seed aging on the Brassica napus seed bacterial community, we conducted accelerated aging tests (45 degrees C, humidity > 95%) with seed lots of four genotypes originating from two field sites in Germany. We found a strong effect of accelerated aging on germination, seedling phenotypes, as well as the seed bacterial community. Control seeds developed mainly into normal seedlings and were characterized by diverse bacterial communities comprising typical core seed microbes. Accelerated aging resulted in abnormal germination and reduced total germination. Furthermore, accelerated aging reduced diversity and evenness of the seed bacterial community and contributed to a shift from Gram-negative to Gram-positive bacteria. This effect, especially the enrichment of Firmicutes, was found irrespective of the genotype and field site; however, the way stress affected bacterial taxa varied, depended on both factors. Tumebacillus and Bacillus showed a significant negative correlation with germination phenotype, whereas alpha diversity correlated positively with a high total germination. At the functional level, the majority of isolated bacteria demonstrated plant-beneficial characteristics, showing a greater beneficial potential in the aged seeds. Our results show that accelerated aging tests affect the seed bacterial community structure and diversity, and correlate with the presence of certain taxa, which might have an effect on germination and seedling phenotype.
The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure–function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3β1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [ 3 H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3β1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.
A major advantage of producing therapeutic proteins in mammalian cells is their ability to tailor proteins with human-like posttranslational modifications such as glycosylation, which ultimately defines aspects like stability, protein folding or immunogenicity. However, producing therapeutic proteins with a consistent and reproducible glycoprofile remains a major challenge for the biopharmaceutical industry, especially with biosimilar production. While the enzymes responsible for glycosylation of proteins have been the subject of various cell engineering approaches, tuning their gene expression to precise levels is still difficult to achieve.While CRISPR/Cas9 enabled the genetic engineering of cells to drastically overexpress or remove a target gene, CRISPR/dCas9-based epigenetic editing by targeted DNA methylation promises to stably change the expression pattern of target genes after transient transfection of the CRISPR-tool. Application of targeted DNA methylation so far has mostly been used to completely silence gene expression by fully methylating the corresponding promoter regions. Here, we aim to tune expression of the associated gene by DNA methylation of confined promoter regions and to apply this technique as a new glycoengineering approach.By coupling CRISPR-based targeted DNA methylation with lectin-FACS assisted sorting we obtained CHO cell lines with a fine-tuned phenotype. First, dCas9-DNMT3A3L in combination with one single gRNA is targeted to the FUT8 promoter to induce confined DNA methylation, resulting in a phenotypically diversified population. Next, a window sorting strategy based on lectin-stained cells using five different sorting gates spanning from low to high FUT8 expression was applied to isolate single clones with a defined phenotype. Isolated clones were phenotypically assessed and re-sorted to obtain a homogenous expression profile. The resulting clonal cell lines showed either tuned or knock-down phenotypes with varying gene expression levels. Two out of seven clones that showed tuned FUT8 gene expression were phenotypically stable over 60 days. Gene expression levels, on the other hand, showed a steady decline over time that in part, however, can be explained by the general variation of FUT8 expression in different growth phases. Importantly, glycan analysis of recombinant EpoFc produced in the tuned clonal cell lines showed ranges of 35-70% fucosylation, demonstrating that isolated clones can produce recombinant proteins with a distinct glycosylation profile. To understand why some clones showed tuned FUT8 gene expression levels while others were completely knocked-down, we analyzed the DNA methylation status of their respective FUT8 promoter. Critical areas within the FUT8 promoter were identified, with some associated with general repression and others with the tuning of FUT8 gene expression when affected by DNA methylation. Additionally, a combination of histone marks associated with active and repressed promoters was found to potentially define clones with a fine-tuned expression. Combined, the data demonstrates that using targeted DNA methylation in a manner confined to specific promoter regions opens new engineering strategies to fine-tune gene expression in mammalian cells.
Background Ethanol (EtOH) represents a promising carbon source for microbial fermentations, aimed at sustainable production of recombinant proteins for food and agricultural applications. However, EtOH-regulated expression systems in Komagataella phaffii remain poorly characterized. To address this gap, we investigated the regulation of promoters within the EtOH utilization (EUT) pathway i.e. alcohol dehydrogenase 2 and 900 (P ADH2 /P ADH900 ), aldehyde dehydrogenase 4 (P ALD4 ) and acetyl-CoA synthetase 1 and 2 (P ACS1 /P ACS2 ). Results We analyzed the activities of EUT promoters in wild-type and knock strains lacking ADH2 and ADH900 across different carbon sources, measuring reporter protein levels and resulting extracellular metabolite profiles. Δ adh2 and Δ adh2 + Δ adh900 strains showed impaired growth on EtOH, while Δ adh900 displayed slightly reduced growth. The wild-type produced 2.0 ± 0.9 g L⁻¹ EtOH on 1% glucose and Δ adh900 strain still produced up to 1.5 ± 0.5 g L -1 . EtOH feeding experiments showed that 1% EtOH in minimal medium optimally supported eGFP expression, whereas higher concentrations (up to 12%) limited both growth and expression. Independent of carbon source, P ADH2 -driven eGFP expression was similar in the wild-type, yet eGFP produced per cell increased in knockout strains. P ALD4 was upregulated in Δ adh2 mutants during growth on glucose while P ACS1/2 and P ALD4 were upregulated in Δ adh900 strains grown on EtOH. P ADH900 was repressed and P ACS1 promoter was upregulated by EtOH. Conclusion ADH2 is primarily responsible for EtOH consumption, while ADH900 contributes to EtOH production, though single knockouts revealed a functional overlap: Δ adh900 still produced EtOH, and Δ adh2 displayed minimal growth on EtOH. In both single knockouts, residual EtOH levels remained on glucose. Our findings give new insights on the regulation of EUT promoters and their role in K. phaffii ’s EtOH metabolism: Δ adh2 and Δ adh900 cells rendered P ADH2 -driven expression to be more EtOH-responsive than in the wild-type due to impaired EtOH consumption leading to more stable EtOH levels. P ALD4 likely responds to accumulating EtOH in Δ adh2 on glucose while P ACS1/2 and P ALD4 are upregulated in Δ adh900 on EtOH, assumingly due to elevated acetaldehyde or acetate levels with blocked reduction of acetaldehyde to EtOH.
The oxidative formation of AcCoA limits the glycolytic pathway yield (YPGLY) for citric acid due to the NADH overflow and carbon loss as CO2. An interesting approach to enhance product yields is the incorporation of carbon-conserving pathways. This study assesses the potential of a carbon-conserving AcCoA pathway, the glycolysis alternative high carbon yield cycle (GATHCYC), to improve citric acid production, utilizing the nonnative citric acid producer Komagataella phaffii as an orthogonal test system. The combination of different metabolic engineering strategies enabled K. phaffii to acquire the ability to produce extracellular citric acid. By constructing the GATHCYC in the cytosol and peroxisomes, the intracellular concentration of AcCoA increased. Overexpression of the genes encoding pyruvate carboxylase (PYC2), citrate synthase (CIT2) and citrate exporter protein (cexA) in the peroxisomal AcCoA strains boosted the citric acid production. Thus, the best producer strain reached a citric acid titer of 51.3 +/- 0.9 g L- 1 and a yield of 0.59 +/- 0.01 g g- 1 after 76 h of glucose-limited fedbatch cultivation. Our results highlight the potential of using GATHCYC to provide an efficient supply of acetylCoA to enhance citric acid production. This approach could be exploited for the production of other AcCoAderived compounds of industrial relevance in different cell factories.
