Microbial Cell Factories最新文献

Background: Single-cell protein (SCP) is gaining attention as a source of food and feed, offering a lower environmental footprint than traditional agriculture. Efficient SCP production requires high cell density culturing (HCDC; >20 g cell dry weight L^- 1), but O₂ supply can become limiting in conventional aerobic systems. To circumvent this bottleneck, we recently proposed an anaerobic strategy using nitrate as an electron acceptor, exploiting denitrification-driven alkalinization in a pH-stat system to regulate substrate provision.

Results: Using the model organisms Paracoccus denitrificans and Paracoccus pantotrophus, we achieved biomass concentrations of up to 60 g dry weight L^- 1 and protein contents up to 75% (75 ± 5%) in a 3 L fed-batch bioreactor. However, growth rates at high cell density were markedly lower than µ_max observed in low-density cultures. In vivo and in silico experiments revealed three interacting constraints: (i) a CO₂-pH lag where CO₂ accumulation delayed alkalization by denitrification and thus substrate injection; (ii) mixing limitations, leading to poor substrate distribution; and (iii) physiological stress from nitrite accumulating during imbalanced denitrification. The CO₂-pH lag emerged as the dominant barrier, resulting in long starvation periods. Lowering the pH setpoint of the pH-stat accelerated CO₂ removal and thus substrate provision, but intensified nitrite toxicity. Insufficient mixing compounded the growth limitations as nitrate was only briefly available to a fraction of the population. Small-batch bioassays ruled out accumulation of inhibiting compounds other than nitrite. However, cells grown at high density in the reactor displayed reduced respiration rates, suggesting chronic stress under these conditions.

Conclusions: Anaerobic HCDC by denitrification is feasible and yields high-quality biomass, but barriers remain to achieving competitive production rates. The CO₂-pH lag appears to be the primary constraint, amplified by incomplete mixing and nitrite toxicity. These factors interact, e.g. mitigating the CO₂-pH lag by lowering the process pH exacerbates nitrite toxicity. Future work should integrate reactor engineering to improve mixing and gas removal and strain selection for tolerance to low pH and nitrite, supported by omics and metabolic modelling to understand denitrifier physiology at high cell density.

Background: The marine diatom Phaeodactylum tricornutum is a promising platform for the sustainable production of terpenoids. This is due to its robust photosynthetic growth and natural accumulation of terpenoids, mainly including photosynthetic pigments. P. tricornutum harbors the methyl-erythritol-phosphate (MEP) pathway in the chloroplast, a dedicated route used for the production of terpenoid-like photosynthetic pigments. Despite its natural predisposition for terpenoid production in the chloroplast, previously reported titers of heterologously produced terpenoids in P. tricornutum are relatively low.

Results: In this study, we used a metabolic engineering strategy to enhance the production of the terpenoid pinene by increasing the production of their precursors. We episomically co-expressed either an isopentenyl diphosphate isomerase (IDI), a geranyl diphosphate synthase (GPPS), or both, along with a pinene synthase (PinS) in the chloroplast of P. tricornutum. We found that the combination of both IDI and GPPS leads to the hyperaccumulation of the monoterpenoid pinene, compared to the strain with only pinene synthase or IDI and GPPS expressed individually. Furthermore, the integration of all three genes in the genome resulted in a strain with a 92-fold higher pinene production, compared to the strain expressing only the pinene synthase. Lastly, we cultivated one of the high-performing transgenic strains at different light intensity regimes and found that the production of pinene increased at elevated light intensities.

Conclusion: In this study, we performed metabolic engineering in the chloroplast of P. tricornutum by expressing heterologous IDI and GPPS together with a pinene synthase. We showed that this approach considerably boosted pinene production, especially in strains where the genes were randomly integrated in the genome. Moreover, we further increased pinene titers by modulating the light intensity during cultivation. Overall, we demonstrated the potential of combining metabolic engineering with optimized cultivation parameters, specifically light intensity, to enhance the production of monoterpenoids in the chloroplast of P. tricornutum.

The rise of difficult-to-treat fungal infections necessitates novel therapeutic strategies. In this study, endophytic fungi were isolated from Acalypha hispida leaves and molecularly identified as Penicillium oxalicum via 18S rRNA sequencing. LC-MS/MS analysis of the fungal extract revealed major bioactive compounds, including linoleic acid, sinapinic acid, alternariol monomethyl ether, ellagic acid, and kaurenic acid. Oily-core poly (ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) nanocapsules (PEGylated PLGA NCs) were developed to encapsulate the fungal extract, improving stability and bioavailability. The PEGylated PLGA NCs exhibited controlled particle size, positive surface charge, and spherical morphology. In vitro, the PEGylated PLGA NCs demonstrated antifungal activity against Candida albicans with inhibition zones of 10-14 mm. In vivo, treatment significantly improved histological features of the kidney, liver, and spleen, and reduced tumor necrosis factor-alpha and cyclooxygenase-2 expression. In silico studies further confirmed the potential of the major compounds of the fungal extract to inhibit C. albicans aspartic proteinases SAP4-6. These findings suggest that PEGylated PLGA NCs loaded with P. oxalicum extract represent a promising antifungal therapeutic strategy.